Duarte AE et al. (2015), Polyphenolic Composition and Evaluation of Anti...

ECB-ART-44430

Molecules 2015 Dec 29;211:E2. doi: 10.3390/molecules21010002.

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Duarte AE , Waczuk EP , Roversi K , da Silva MA , Barros LM , da Cunha FA , de Menezes IR , da Costa JG , Boligon AA , Ademiluyi AO , Kamdem JP , Rocha JB , Burger ME .

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Raphiodon echinus (R. echinus) is used in Brazilian folk medicine for the treatment of inflammation, coughs, and infectious diseases. However, no information is available on the potential antioxidant, cytotoxicity and genotoxicity of this plant. In this study, the polyphenolic constituents, antioxidant capacity and potential toxic effects of aqueous and ethanolic extracts of R. echinus on human erythrocytes and leukocytes were investigated for the first time. R. echinus extracts showed the presence of Gallic, chlorogenic, caffeic and ellagic acids, rutin, quercitrin and quercetin. Aqueous and ethanolic extracts of R. echinus exhibited antioxidant activity in DPPH radical scavenging with IC50 = 111.9 μg/mL (EtOH extract) and IC50 = 227.9 μg/mL (aqueous extract). The extracts inhibited Fe(2+) (10 μM) induced thiobarbituric acid reactive substances (TBARS) formation in rat brain and liver homogenates. The extracts (30-480 μg/mL) did not induce genotoxicity, cytotoxicity or osmotic fragility in human blood cells. The findings of this present study therefore suggest that the therapeutic effect of R. echinus may be, in part, related to its antioxidant potential. Nevertheless, further in vitro and in vivo studies are required to ascertain the safety margin of its use in folk medicine.

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	Figure 1. HPLC-DAD chromatograms of aqueous (A) and ethanolic (B) extracts of the leaves of Raphiodon echinus (R. echinus): Gallic acid (peak 1), chlorogenic acid (peak 2), caffeic acid (peak 3), ellagic acid (peak 4), rutin (peak 5), quercitrin (peak 6) and quercetin (peak 7). Calibration curve for Gallic acid: y = 12574x + 1307.8 (r = 0.9999); chlorogenic acid: y = 11953x + 1278.2 (r = 0.9995); caffeic acid: y = 11976x + 1187.0 (r = 0.9996); ellagic acid: y = 13169x + 1346.8 (r = 0.9999); quercitrin: y = 12473x + 1187.5 (r = 0.9991); rutin: y = 12814x + 1189.3 (r = 0.9999) and quercetin: y = 12537x + 1375.6 (r = 0.9994). All chromatography operations were carried out at ambient temperature and in triplicate.
	Figure 2. Quenching of DPPH radicals by aqueous and ethanolic extracts from the leaves of R. echinus. Data are expressed as mean ± SEM of n = 4 independent experiments.


	Figure 5. Oxidation of Fe2+ by aqueous (A) and ethanolic (B) extracts from the leaves of R. echinus (1–60 μg/mL). The extracts (30–120 μg/mL) were incubated with FeSO4 (110 μM) for 10 min. Then, ortho-phenanthroline was added and the absorbance of the reaction mixture was measured at 0, 10 and 20 min following its addition. After the last reading (at 20 min), 5 mM ascorbic acid (AA) was added to the reaction mixture, and the absorbance was read again after 5 min (at 25 min), 10 min (at 30 min) and 20 min (at 40 min) (see Table 1 for details). Values represent the mean ± SEM of three independent experiments performed in duplicate. AERE, aqueous extract of R. echinus; EERE, ethanolic extract of R. echinus.

	Figure 7. Osmotic fragility of human erythrocytes treated with aqueous (A) and ethanolic (B) extracts of the leaves of R. echinus. Untreated and treated erythrocytes were added to different concentrations of NaCl (0%–0.9%) and incubated for 20 min. The absorbance of the supernatant was measured and the hemolysis in each tube was expressed as percentage of control. Each bar represents the mean of n = 3 independent experiments performed in triplicate ± SEM.